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Abstract:

A method of manufacturing a substrate is provided. The method comprises,
in some aspects, a) providing a support; b) forming a template by
attaching a plurality of polymeric nanoparticles some or all having a
core-shell structure to the support, wherein the core comprises a first
polymer and the shell comprises a second polymer; and c) forming the
metal nanoarray substrate by attaching a plurality of metallic
nanoparticles to at least some of the polymeric nanoparticles of the
template. A biosensor comprising a substrate manufactured by the method,
and a method for the detection of an analyte in a sample by surface
enhanced Raman spectroscopy (SERS) is also provided.

Claims:

1. A method of manufacturing a metal nanoarray substrate, the method
comprising a) providing a support; b) forming a template by attaching a
plurality of polymeric nanoparticles each having a core-shell structure
to the support, wherein the core comprises a first polymer and the shell
comprises a second polymer; and c) forming the metal nanoarray substrate
by attaching a plurality of metallic nanoparticles to the polymeric
nanoparticles of the template.

2. The method according to claim 1, wherein the plurality of polymeric
nanoparticles is formed by a) copolymerizing the first polymer and the
second polymer to form an amphiphilic copolymer; and b) dispersing the
amphiphilic copolymer in a suitable solvent to form reverse micelles.

3. The method according to claim 1, wherein the template size and
geometry is controlled by controlling the size and geometry of the
polymeric nanoparticles by controlling the molecular weight of the
polymer or the polymeric nanoparticle-forming conditions.

4. The method according to claim 3, wherein control of the polymeric
nanoparticle-forming conditions comprises control of the relative
humidity during polymeric nanoparticle formation.

5. The method according to claim 4, wherein the polymeric nanoparticles
are reverse micelles.

6. The method according to claim 1, wherein the first polymer exhibits a
positive charge in an aqueous medium having a pH of less than about 8.

8. The method according to claim 1, wherein the first polymer comprises
poly(2-vinyl pyridine).

9. The method according to claim 1, wherein the second polymer comprises
a hydrophobic unit.

10. The method according to claim 1, wherein the second polymer is
selected from the group consisting of polystyrene, polyolefin,
polysiloxane, polyvinyl naphthalene, polyvinyl anthracene, and mixtures
thereof.

11. The method according to claim 10, wherein the second polymer
comprises polystyrene.

12. The method according to claim 1, wherein the polymeric nanoparticles
comprises or consists essentially of a block copolymer of polystyrene and
poly(2-vinylpyridine).

13. The method according to claim 1, wherein the plurality of polymeric
nanoparticles forms an array having an average inter-particle distance of
less than 50 nm on the support.

14. The method according to claim 13, wherein the plurality of polymeric
nanoparticles forms an array having an average inter-particle distance of
about 10 nm on the support.

15. The method according to claim 1, wherein the polymeric nanoparticles
attached to the surface are subjected to a treatment to vary the template
size or remove the polymeric template.

17. The method according to claim 1, wherein the metallic nanoparticles
are negatively charged metallic nanoparticles.

18. The method according to claim 1, wherein the metallic nanoparticles
are attached to the polymeric nanoparticles by electrostatic interaction.

19. The method according to claim 1, wherein the metallic nanoparticles
comprise or consist essentially of gold.

20. The method according to claim 19, wherein the metallic nanoparticles
are citrate-stabilized gold nanoparticles.

21. The method according to claim 1, wherein the metallic nanoparticles
attached to the exposed cores of the polymeric nanoparticles have an
inter-particle distance of less than 5 nm.

22. The method according to claim 1, wherein the mean diameter of the
metallic nanoparticles is in the range of about 5 nm to about 15 nm.

23. The method according to claim 1, wherein the polymeric nanoparticles
and/or the metallic nanoparticles are essentially monodisperse.

24. The method according to claim 1, wherein the average number of
metallic nanoparticles on each polymeric nanoparticle is in the range of
about 1 to about 25.

25. The method according to claim 24, wherein the average number of
metallic nanoparticles on each polymeric nanoparticle is about 18.

26. The method according to claim 1, wherein the support comprises a
metallic nanoparticle attached to the surface of the support, wherein the
metallic nanoparticle is formed by first forming a polymeric
nanoparticle, contacting the polymeric nanoparticle with a solution
containing metal ions, and removing the polymer, thereby forming metallic
nanoparticles in situ.

27. The method according to claim 26, wherein the metallic nanoparticle
is a gold nanoparticle.

28. The method according to claim 26, wherein the polymeric nanoparticle
comprises or consists essentially of a block copolymer of polystyrene and
poly(2-vinylpyridine).

29. The method according to claim 26, wherein the solution containing
metal ions is an aqueous solution containing gold ions.

30. The method according to claim 26, wherein the polymer is removed by
reactive ion etching.

31. The method according to claim 26, wherein the formation of the
template is carried out by attaching a plurality of polymeric
nanoparticles each having a core-shell structure to the metallic
nanoparticles attached to the surface of the support.

32. The method according to claim 31, wherein forming the metal nanoarray
comprises attaching a plurality of metallic nanoparticles to the
polymeric nanoparticles of the template and the metallic nanoparticles
attached to the surface of the support.

33. The method according to claim 1, wherein the formation of the
template is carried out by attaching a plurality of polymeric
nanoparticles each having a core-shell structure directly to the surface
of the support.

34. The method according to claim 1, wherein the surface of the support
where the plurality of polymeric nanoparticles is attached to is
non-planar.

35. The method according to claim 1, wherein the support comprises an
optical fiber.

36. The method according to claim 35, wherein the plurality of polymeric
nanoparticles is attached to the optical fiber by drop coating.

37. The method according to claim 1, wherein the first polymer exhibits
an electric charge when present in an aqueous solution.

38. A metal nanoarray substrate obtained by the method of claim 1.

39. A metal nanoarray substrate obtained by the method of claim 32.

40. A biosensor comprising a metal nanoarray substrate manufactured by a
method according to claim 1.

41. A method for the detection of an analyte in a sample by SERS,
comprising contacting the sample with the biosensor according to claim
40.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application makes reference to and claims the benefit of
priority of an application for "A Method For Fabricating Metal Nanoarrays
On Optical Fiber Faucet For High-Performance SERS Based Remote Sensing Of
Molecular Analytes, Using Directed Self-Assembly Gold Nanoparticles"
filed on Aug. 19, 2011, with the Intellectual Property Office of
Singapore, and there duly assigned serial number 201106015-9. The content
of said application filed on Aug. 19, 2011, is incorporated herein by
reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Some aspects of the invention relate to methods of forming
substrates for optical sensing by surface enhanced Raman spectroscopy
(SERS), and to substrates formed by the methods.

BACKGROUND

[0003] Vibrational spectroscopic techniques, such as infra-red (IR),
normal Raman Spectroscopy and Surface Enhanced Raman Spectroscopy (SERS),
have been considered for analyte detection. Of these, SERS has evolved as
one of the more sensitive techniques for analyte detection due to the
enhancement of the Raman spectral intensity by interaction of the
adsorbed SERS active analyte molecules with the surface of a metal
substrate.

[0004] A major application for SERS substrates is in its use as a
biosensor. With an extremely small cross-sectional Raman scattering area
of 10-29 cm2, Raman scattering signals are innately weak.
Contrary to previously held presumptions that laser excitation frequency
forms the basis for signal enhancement, the density of Raman hotspots on
a substrate surface is presently considered to be an important factor
affecting Raman signal intensity. For SERS substrates comprising
nanoparticles, for example, a Raman hotspot can exist in a gap or
junction between adjacent metal nanoparticles that are in close proximity
to one other. These hotspots have been identified using atomic force
microscopy (AFM) characterization and SERS studies as chemisorptions site
for analyte molecules. Near convergence of two nanoparticles may induce
coupling of their individual transition dipoles, which include ballistic
carriers in oscillation. Coherent interference of their electromagnetic
(EM) field may lead to a red-shift in coupled plasmon resonance, and may
result in amplification of the signal intensity. Accordingly, the
strength of the Raman signal has been found to be proportional to the
number of hotspots. By varying the density of Raman hotspots on a SERS
substrate, signal enhancement of up to 14 orders in magnitude has been
reported.

[0005] Although SERS has established itself as an important analytical
technique in recent years, there remains a need for substrate-related
improvements for wider adoption of the technique in biological and
environmental sensing. Commercialization of SERS techniques has thus far
been limited due to a number of challenges.

[0006] Firstly, to achieve effective biosensing capability, the inherently
large variation of Raman signals has to be ameliorated. As the SERS
substrate forms a key component in SERS measurements, various groups have
attempted to provide improved SERS substrates. Generally, a good SERS
substrate should be capable of producing optimal Raman signal enhancement
with reliable reproducibility. However, state-of-the-art SERS substrates
often suffer from non-uniform enhancement across their surfaces, as
existing substrate fabrication processes aim to enhance signals for
single-molecule detection, and as a result, produce hotspot congregations
that are highly localized. For practical applications, substrates with
high reproducibility are more suitable as they allow consistent
generation of SERS results.

[0007] Other substrate-related issues include inconsistent signal
enhancement at different points on the same substrate, batch-to-batch
variations in signal, the complexity of fabrication, cost effectiveness
of mass production, the stability of the substrate, and the difficulty of
detecting wide range of analytes.

[0008] Even though techniques such as electron beam lithography have been
used to produce precise and well-defined metallic arrays on substrates to
overcome such reproducibility issues, these techniques are expensive and
time consuming. Furthermore, these techniques lack the ability to
fabricate arrays over macroscopic areas, thereby posing problems in terms
of scalability. State-of-the-art techniques are also usually not
versatile, in that they are not able to be used on the surfaces of some
types of material, and are not able to be used on non-planar surfaces.

[0009] In one specific SERS technique, optical fibers have been used for
in situ monitoring. This technique has various advantages over
conventional substrate-based methods, such as compactness, flexibility
and remote sensing capability. Therefore, cost-effective and reliable
substrate fabrication techniques that may be extended to optical fibers
hold great value in taking SERS-based sensing into practical utility in a
number of areas, e.g. the bio-processing industry, real-time monitoring
of chemical reactions and in vivo biosensing, and monitoring of toxic
chemical/biological warfare agents.

[0010] Conventional two-dimensional arrays of gold or silver nanoparticles
have been achieved on optical fibers using self-assembly of gold
nanoparticles on amine or thiol terminated silane self assembled
monolayers (SAMs). However, inconsistencies due to the lack of
reproducibility of the self-assembly process, low signal enhancement in
SERS, and the possibility of random multilayer formation on the fiber
tip, resulting in opaque fiber faucets, are issues compromising the
applicability of this technique.

[0011] Even though techniques such as UV lithography and nanoimprinting
have also been used, these techniques still suffer from limitations
relating to signal enhancement, as well as ease of fabrication. To
achieve higher signal enhancement, researchers have used an optical fiber
with a SERS substrate at the tip in a metal nanoparticle solution to
increase the number of hotspots. However, this technique is cumbersome
and cannot be translated into biological environment, since nanoparticle
solutions cannot survive the harsh ionic conditions of biological media.

[0012] In view of the above, there remains a need for an improved
substrate for optical sensing using SERS, as well as improved methods for
forming the substrate that addresses at least one or more of the
above-mentioned problems.

SUMMARY OF THE INVENTION

[0013] In a first aspect, the invention refers to a method of
manufacturing a metal nanoarray substrate. The method comprises:

[0014]
a) providing a support;

[0015] b) forming a template by attaching a
plurality of polymeric nanoparticles some or all having a core-shell
structure to the support, wherein the core comprises a first polymer and
the shell comprises a second polymer; and

[0016] c) forming the metal
nanoarray by attaching a plurality of metallic nanoparticles to at least
some of the polymeric nanoparticles of the template.

[0017] In a second aspect, the invention refers to a metal nanoarray
substrate obtained by an inventive method according to the first aspect.

[0018] In a third aspect, the invention refers to a biosensor comprising a
metal nanoarray substrate manufactured by an inventive method according
to the first aspect.

[0019] In a fourth aspect, the invention refers to a method for the
detection of an analyte in a sample by SERS, comprising contacting the
sample with the biosensor according to the second aspect.

[0020] In addition, other aspects of the invention are discussed in more
detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Various aspects of the invention will be better understood with
reference to the detailed description when considered in conjunction with
the non-limiting examples and the accompanying drawings, in which:

[0022] FIG. 1A is a schematic diagram showing a general procedure to
manufacture a metal nanoarray substrate according to various aspects of
the invention. As shown in (i), a cross-sectional view of support 105 is
provided. A template is formed by attaching a plurality of polymeric
nanoparticles, each having a core-shell structure, wherein the core
comprises a first polymer 101 and the shell comprises a second polymer
103, to a surface of the support 105. In various embodiments, the
polymeric nanoparticles are present as discrete particles on the surface
of the support. In (ii), the plurality of polymeric nanoparticles is
subjected to an optional treatment step to control or to vary the size of
the polymeric nanoparticles. As shown in (iii), the metal nanoarray
substrate is formed by contacting the polymeric nanoparticles with
metallic nanoparticles 107, such that the metallic nanoparticles 107 are
attached to the polymeric nanoparticles of the template as shown in (iv).

[0023] FIG. 1B is a schematic diagram showing a procedure to manufacture a
metal nanoarray substrate according to certain aspects of the invention.
As depicted in the figure, a metal nanoarray substrate comprising gold
nanoparticle cluster arrays on a silicon (Si) or glass support is
prepared. In the embodiment shown, polymeric nanoparticles comprising the
block copolymer polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) is used.
The polymeric nanoparticles that are attached to the support function as
a template to fabricate the gold nanoparticle cluster arrays on the
support. In (i), a cross-sectional view of the hemispherical profiles of
the polymeric nanoparticles on a support comprising or consisting
essentially of silicon or glass is depicted. The polymeric nanoparticles
having a core-shell structure, wherein the core comprises a first
polymer, poly(2-vinylpyridine), and the shell comprises a second polymer,
polystyrene, are attached to a surface of the support to form the
template. In the embodiment shown, the plurality of polymeric
nanoparticles is also subjected to a treatment in the form of a
controlled oxygen (O2) plasma reactive ion etch (RIE) to vary the
template size. Depending on the time of treatment and thickness of the PS
shell, for example, the RIE removes some of the PS shell, and may expose
the poly(2-vinylpyridine) core. Accordingly, the size of the polymeric
nanoparticles is reduced. In (iii), the polymeric nanoparticles are
incubated in a solution comprising citrate-stabilized colloidal gold
nanoparticles at a pH of 5.8. The pH of the solution is less than the
isoelectric point (pI) of 8.3 for the PS-b-P2VP nanoparticles. The inset
of the figure shows the electrostatic attraction experienced by the
negatively charged gold (Au) nanoparticles to the positively charged
polymeric nanoparticles comprising the P2VP core. The Au nanoparticles
cluster tightly on the polymeric nanoparticles that are attached to the
support to form the metal nanoarray substrate as shown in (iv). The size
of the Au nanoparticle clusters may be determined by the feature size,
for example, the size of the polymeric nanoparticles and the separation
between the polymeric nanoparticles.

[0024] FIG. 2 is a graph showing variation in zeta potential of
polystyrene-block-poly(2-vinylpyridine) (PS-b-PVP) thin films with the pH
of the solution. The isoelectric point (pI) of 8.3 is indicated. Also
indicated is the zeta potential of 30.6 mV at a pH of 5.8, which is the
pH of the citrate-stabilized gold nanoparticle solution.

[0025] FIGS. 3 (a) to (h) are plan-view transmission electron microscopy
(TEM) images. FIGS. 3 (a) to (d) are TEM images of measurements taken at
high magnifications. FIGS. 3 (e) to (h) are TEM images of measurements
taken at low magnifications. The images show gold nanoparticle clusters
with increasing number of particles per cluster. The histrograms of the
particle numbers per cluster (N) are shown in FIG. 4 (a). The scale bars
in FIGS. 3 (a) to (d) depict a length of 100 nm, while those in FIGS. 3
(e) to (h) depict a length of 200 nm.

[0026] FIG. 4 (a) are graphs depicting histograms of the number of
particles per cluster for four different template sizes, where N=5, 8, 13
and 18. The average number of particles per cluster (N) is shown against
each histogram. FIG. 4 (b) is a photograph showing samples of
nanoparticle cluster arrays obtained on a glass chip. As can be seen from
the photograph, there is variation in hue across the samples, which may
be attributed to changes in cluster size. Uniformity across the coated
area of the chip is readily discernible from the photograph.

[0027] FIG. 5 (a) is a graph showing variation of the number of
nanoparticles per cluster (N), as a function of height (R) of the
templates. The data is fitted with a quadratic curve of the form
y=Cx2. In so doing, and in comparing with Equation (c) (see below),
the value of C is 0.027. FIG. 5 (b) is a schematic diagram depicting the
3D nature of the gold nanoclusters on the curved hemispherical template
with a radius of R. FIG. 5 (c) is a schematic diagram showing
characteristic length scales between a pair of nanoparticles in a
cluster. The radius of the nanoparticle is denoted as r. The effective
radius reff represents inter-particle separation caused by repulsive
interaction due to the presence of negatively charged citrate ligands.

[0028] FIGS. 6 (a) and (b) are cross-section and top-view schematic
diagrams depicting geometric parameters of (a) templates; and (b)
nanoparticle clusters. FIG. 6 (c) is a schematic diagram illustrating the
calculation of edge-edge separation between the templates and the
clusters from their distributions in the respective geometric parameters
(viz. R, d and P). In the figures, P=periodicity of lattice; R=radius of
the template; d=2r=diameter of Au nanoparticles (NP);
St=inter-template separation; and Sd=inter-cluster separation.

[0029] FIG. 7 are tapping mode atomic force microscope (AFM) images of:
(a) and (b) templates; (c) and (d) templates with nanoparticles. (a) and
(c) are measured under low resolution and (b) and (d) are measured under
high resolution. In the images shown, there are systematically increasing
separations from left to right. The scale bars in (a) and (c) depict a
length of 400 nm, while those in (b) and (d) depict a length of 100 nm.

[0030] FIG. 8 are graphs showing extinction spectra of gold nanoparticle
cluster arrays with (a) increasing values of N, where N=5, 8, 12 and 18
(a spectrum of isolated gold nanoparticles is also included for
reference); and (c) decreasing values of separation, where the separation
is 37 nm, 30 nm, 22 nm and 10 nm (their corresponding curves are shown in
direction of the arrow).

[0032] FIGS. 10 (a) and (c) are graphs showing SERS signal intensity for
the major peaks of CV molecules comparing intensity enhancement with (a)
increases in cluster size N, with N=5, 8, 13 and 18 (arranged in
decreasing order from N=18 to N=5, from left to right in the graph), and
(b) decreases in cluster separation, where the separation is 37 nm, 30
nm, 22 nm, and 10 nm (arranged in decreasing order from separation=37 nm
to separation=10 nm, from left to right in the graph). FIGS. 10 (b) and
(d) are graphs comparing intensity and corresponding SERS enhancement
factors (EF) of the most intense peak for CV as a function of (b) cluster
size; and (d) separation.

[0033] FIG. 11 is a bar graph comparing the signal intensity for the most
intense peak of the CV molecule obtained on (b) cluster arrays with N=18
and a separation of 10 nm, versus (a) unpatterned gold nanoparticles on
silicon substrates ("unpatterned control"), and (c) commercial available
Klarite® (Renishaw Diagnostics) substrates as controls. The
unpatterned control was obtained by adsorption of citrate stabilized gold
nanoparticles on aminosilane treated silicon substrates. As can be seen
from the figure, there is a clear increase in SERS performance of the
clusters as compared with the controls.

[0034] FIG. 12 (a) is a tapping mode atomic force microscope (AFM) image
of the template deposited by drop-coating on the tip of a polished
optical fiber. The conformal deposition of the reverse micelles on the
rough asperities of the surface of the fiber tip is clearly discernable.
The scale bar in FIG. 12 (a) denotes a length of 400 nm. FIG. 12 (b) is
an optical photograph of the fiber tip covered with gold nanoparticle
cluster arrays. The scale bar in FIG. 12 (b) denotes a length of 200
μm. FIG. 12 (c) is an optical photograph showing the area where
reflectance spectrum was collected. A microspectrometer measuring a spot
of 77 μm×77 μm was used. The scale bar in FIG. 12 (c) denotes
a length of 100 μm. FIG. 12 (d) is a graph showing the reflectance
spectrum having a plasmonic peak at a wavelength of about 640 nm.

[0035] FIG. 13 (a) is a schematic diagram showing the measurement
configuration used for measuring SERS on a fiber. FIGS. 13 (b) and (c)
are optical photographs showing a measurement set-up that was used to
collect signal from a CV solution from one end of the optical fiber, and
measured at other. FIG. 13 (c) shows the fiber faucet covered with gold
nanoparticle clusters dipped within the CV solution within a vial, while
the other end faces the objective lens above.

[0036] FIG. 14 are graphs comparing the SERS signal intensity measured
under (a) direct configuration; and (b) indirect configuration for the
nanoparticle cluster arrays versus unpatterned controls. The unpatterned
control includes isolated nanoparticles obtained by electrostatic
adsorption of gold nanoparticles to aminosilane treated fiber. The direct
measurement configuration measures SERS under backscattering geometry on
the fiber tip surface incubated in CV solution overnight. The indirect
configuration corresponds to SERS measurements performed through fiber,
with the cluster-containing end dipped in solution and the other end
facing the objective. FIGS. 14 (c) and (d) are graphs comparing between
the most intense peak of the CV molecule obtained in FIGS. 14 (a) and (b)
respectively.

[0038] FIG. 16 is a graph showing a systematic reduction in size of the
template height with increase in RIE duration. Using the slope of the
linear fit line, the etch rate was found to be 19.2 nm.

[0039] FIG. 17 (a) to (d) are tapping mode atomic force microscopy (AFM)
images of templates with systematically increasing heights from (a) to
(d). The templates are obtained from (a) 38 s; (b) 30 s; (c) 22 s; and
(d) Os of oxygen (O2) plasma reactive ion etch (RIE) treatment on
the surface of PS-b-P2VP films. The curves show the size distribution of
the templates, obtained by Gaussian fits made to histograms of heights
obtained from AFM measurements. The scale bar in the figures denotes a
length of 100 nm.

[0040] FIG. 18 (a) to (d) are field emission scanning electron microscopy
(FESEM) images of gold nanoparticle cluster arrays obtained using
different template dimensions realized through controlled O2 plasma
RIE durations as indicated in the figures. The scale bar in the figure
denotes a length of 200 nm.

[0041] FIG. 19 (a) is a block diagram illustrating the steps used for
extraction of 3D coordinates using TEM plan view images of the
nanoparticle clusters. FIG. 19 (b) is a series of images of (i) plan-view
TEM image of nanoparticle clusters where N is about 13; (ii) plan-view
TEM image of a single cluster arbitrarily chosen and sectioned; and (iii)
image of (ii) thresholding to subtract background. The (x, y) coordinates
of each Au nanoparticle is obtained with respect to the chosen origin,
with the distances known through magnification of the TEM image. FIG. 19
(b) (iv) is an image depicting planes with different z heights of
isolated nanoparticles obtained by background subtraction. Each sectioned
nanoparticle template is then uploaded in the finite difference time
domain (FDTD) simulation layout that contains polystyrene hemispheres on
the silicon substrate. The z coordinate of the particle is computed from
the point of intersection of the gold nanoparticle and the polystyrene
hemisphere. The FDTD layout made from the extracted (x, y, z) data of
each nanoparticle in sectioned cluster is illustrated to the right in
FIG. 19 (c).

[0042] FIG. 20 (a) to (d) are graphs depicting simulated extinction
spectra for different cluster sizes (N), where (a) N has a value of about
5; (b) N has a value of about 8; (c) N has a value of about 13; and (d) N
has a value of about 18. The separation values are respectively (a) 61.0
nm; (b) 53.3 nm, (c) 45.5 nm and (d) 33.7 nm.

[0043] FIG. 21 is a graph showing a simulated extinction spectrum of N
having a value of about 18 clusters, after removing the periodic boundary
condition showing that the sharp feature about 450 nm appearing in the
periodic clusters is absent. In addition, a contribution around 520 nm
appears, along with a weak modulation around about 650 nm.

[0044] FIG. 22 are E-field profiles of (A) 3D clusters; and (B) imaginary
planar clusters performed for the case where N is about 18. The
simulation shows that the 3D clusters exhibit an E-field enhancement
across a wide area spanning the entire inter-cluster region. This is
however found to be absent in the planar clusters. The scale bar in the
images denotes a length of 10 nm.

[0045] FIG. 23 is a graph depicting the simulated extinction spectra of
N-18 clusters, obtained by modeling Au nanoparticles that are 50%
immersed within the polystyrene template. The simulated spectrum (as
shown in FIG. 24) shows a dominant contribution at 530 nm due to
individual excitation of Au particles, but the peak around 600 nm (as
observed in the clusters) due to plasmonic coupling between nanoparticles
is absent.

[0046] FIG. 24 is a graph depicting the SERS spectra of CV acquired at 12
different locations spaced at least 3 mm apart on substrate, which are
overlaid to show the signal intensity variation. The most intense peak of
CV at 1617 cm-1 is used to compute the error, which in this case is
8.5%.

[0047] FIGS. 25 (a) and (b) are graphs comparing the SERS signal intensity
for the most intense peak of CV of the nanoparticle cluster arrays with
systematic variation in (a) cluster sizes and (b) inter-cluster
separations against unpatterned gold nanoparticle monolayer as control.

[0048] FIG. 26 is a graph depicting the SERS spectra of (b) CV molecule on
cluster array substrates compared against (a) unpatterned gold monolayer
used as a control.

[0049] FIG. 27 is a schematic diagram illustrating the divergent
electrical field emanating from the nanoparticle clusters expected due to
the curved geometry.

[0050] FIG. 28 are graphs showing (a) systematic variation in curvature
(or height (h) to radius (R) ratio) of the reverse micelle templates on
surface, as a function of relative humidity of the environment during
thin film formation; (b) systematic variation in the plasmon resonance of
nanoparticle cluster arrays with variations in the h/R ratio. The
fine-tunability in curvature is a possibility that arises with a
composite core-shell system such as the reverse micelles. The change
arises due to the possible increase in surface tension at the interface
of polystyrene and polyvinyl pyridine, due to absorption of moisture
within PVP, to the extent there is moisture content in the surrounding
environment during the formation of templates on surface. Such
fine-tunability in curvature is found to yield tunability in plasmon
resonance, and it is a significant capability of interest to achieve
higher SERS performance of the resulting arrays. There is a possibility
of fine-tuning the plasmon resonance close to the molecular absorbance,
and the laser excitation wavelength used in order to realize high SERS
enhancements.

[0051] FIG. 29 depicts super-cluster arrays of gold (Au) nanoparticle
clusters with systematically varying structural composition achieved
through control over deposition conditions, and by varying the relative
humidity during spin-coating. `A` refers to in situ prepared Au
nanoparticles obtained from polystyrene-block-poly(2-vinylpyridine) with
a molecular weight of 380 kDa, with fPS˜0.5. `B` refers to
reverse micelle templates formed out of PS-b-P2VP 114 kDa and
fPS˜0.5. `C` refers to citrate-stabilized Au nanoparticles
adsorbed from solution phase. The scale bar in the figures denotes a
length of 100 nm.

[0052] FIG. 30 are graphs showing removal of the supporting polymer
template results in higher SERS enhancements as could be expected due to
a closer separation between nanoparticles, contrary to earlier beliefs
that the nanoparticles formed a fused mass by coalescing together. All
spectra were recorded under identical conditions of probe molecule
deposition, laser excitation wavelength, exposure duration and laser
power. The SERS enhancement was found to distinctly increase upon removal
of the polymer template as shown in FIG. 30 (a). The enhancement further
increased upon formation of a super-cluster (with polymer removed), as
shown in FIG. 30 (b). The influence on the SERS enhancement due to
super-cluster formation may have contributions from the central gold
nanoparticle template, along with the complex plasmonic coupling due to
the super-cluster geometry. The scale bar in the figure denotes a length
of 100 nm.

DETAILED DESCRIPTION OF THE INVENTION

[0053] In a first aspect, the present invention refers to a method of
manufacturing a metal nanoarray substrate. The method comprises, in some
embodiments, providing a support; forming a template by attaching a
plurality of polymeric nanoparticles some or all having a core-shell
structure to the support, wherein the core comprises a first polymer and
the shell comprises a second polymer; and forming the metal nanoarray
substrate by attaching a plurality of metallic nanoparticles to at least
some of the polymeric nanoparticles of the template.

[0054] Through methods such as the first aspect of the invention, a
substrate that may be used for optical sending by surface enhanced Raman
spectroscopy (SERS) is obtained. By attaching a plurality of polymeric
nanoparticles having a core-shell configuration on a suitable support, a
template for subsequent attachment of metallic nanoparticles may be
formed. In various embodiments, the core of the polymeric nanoparticles
comprises or is formed from a first polymer that exhibits an electric
charge when present in an aqueous solution. Depending on the type of the
first polymer and/or the pH of the solution, for example, the charge on
the first polymer may be positive or negative. In various embodiments,
the core of the polymeric nanoparticles comprises or is formed from a
first polymer having an isoelectric point that is higher than the pH of
the solution comprising the metallic nanoparticles. By placing the
support in a solution having a pH that is lower than the isoelectric
point of the first polymer, the polymeric nanoparticles, which are
attached to the support, may attain a positive charge. Subsequently, when
metallic nanoparticles, for example, negatively charged metallic
nanoparticles such as citrate-stabilized gold nanoparticles, are brought
into contact with the polymeric nanoparticles, the metallic nanoparticles
may be attracted to the polymeric nanoparticles, and may be attached to
the polymeric nanoparticles by electrostatic interaction.

[0055] An advantage of a substrate formed by such methods is that no
lithography or electron beam lithography is involved, thus providing a
simple, inexpensive and quick technique to achieve a highly sensitive and
spatially uniform SERS signal, e.g., for biomedical applications. A
treatment step, such as wet etching or dry etching, may be used to treat
the polymeric nanoparticles that are attached to the surface of the
support to vary the template size. In so doing, the resolution of the
template, such as the size of the polymeric nanoparticles and the
interparticle distance between the polymeric nanoparticles, may be
customized in a simple manner. Using such methods, highly uniform,
precise and well-defined metallic arrays of sub-5 nm separations may be
achieved. Furthermore, such methods to form the substrate may be
advantageous in that no linkers are required to attach the metallic
nanoparticles to the polymeric nanoparticles attached on the support.
Instead, the metallic nanoparticles may directly attach to the polymeric
nanoparticles by electrostatic attraction, thereby eliminating extra
process steps. In further embodiments, the treatment step may be used to
remove the polymeric template. In so doing, a metal nanoarray substrate
of arrays of metallic nanoparticles clusters (termed herein as
"super-clusters") on a support may be formed.

[0056] A substrate for optical sensing by SERS, herein also termed a SERS
substrate, generally refers to an engineered metallic nanostructure on
which analyte molecules are adsorbed for SERS acquisitions. Various
embodiments of the present invention relate to a SERS substrate that
provides a highly uniform and reproducible bioanalysis surface.

[0057] Generally, a SERS substrate includes a support having a roughened
metal surface, in which the degree of roughness of the metal surface is
sufficient to induce the SERS effect. The degree of roughness of the
metal surface may result in a reproducible and uniform SERS signal, such
as within about 10% reproducibility error variation over a substrate area
of 1 cm2, for analysis of materials bound to the metal surface of
the substrate. In various embodiments, the SERS signal intensity
variations are as low as 10%.

[0058] Such methods may comprise providing a support. The support used to
form the SERS substrate may generally be formed from any material.
Examples of material that may be used to form the SERS substrate include,
but are not limited to, silicon, glass, ceramic and organic polymers. In
some embodiments, the support is silicon or glass.

[0059] Such methods may comprise forming a template by attaching a
plurality of polymeric nanoparticles having a core-shell structure to a
surface of the support. A "nanoparticle" refers to a particle having a
characteristic length, such as diameter, in the range of up to 100 nm.
The term "polymeric nanoparticles" refers to nanoparticles that comprise
one or more different polymers. The term "plurality" as used herein means
more than one, such as at least 2, 20, 50, 100, 1000, 10000, 100000,
1000000, 10000000 or even more.

[0060] The plurality of polymeric nanoparticles that is attached to the
support surface functions as a template for subsequent attachment of a
plurality of metallic nanoparticles on the polymeric nanoparticles. Using
such methods, it has been found that the templates formed, for example,
on a silicon wafer support, may be highly uniform, e.g., with standard
deviations in the mean values for feature heights, and spacing of less
than 10% (as measured by AFM).

[0061] The template size and geometry may be controlled by controlling the
size and geometry of the polymeric nanoparticles by controlling the
molecular weight of the polymer or the polymeric nanoparticle-forming
conditions. For example, control of the polymeric nanoparticle-forming
conditions may comprise control of the relative humidity during polymeric
nanoparticle formation. In various embodiments, the relative humidity
during polymeric nanoparticle formation is controlled to be in the range
of between about 10% to about 90%, such as about 10% to about 50%, about
10% to about 30%, about 50% to about 90%, or about 30% to about 50%.

[0062] The plurality of polymeric nanoparticles may have a core-shell
structure. The core of the core-shell polymeric nanoparticles may
comprise a first polymer, and the shell of the polymeric nanoparticles
may comprise a second polymer.

[0063] To form the core-shell polymeric nanoparticles, in various
embodiments, a first polymer is used to form nanoparticles, followed by
deposition of a second polymer on the nanoparticles. In so doing, a
plurality of polymeric particles having a core-shell structure, wherein
the core comprises the first polymer and the shell comprises the second
polymer, may be formed. Nanoparticles comprising the first polymer may be
formed, for example, by dispersing the first polymer in a suitable medium
to form an emulsion containing nanoparticles of the first polymer. By
coating the second polymer on the nanoparticles, such as by immersing the
nanoparticles in a solution comprising monomers of the second polymer,
followed by polymerization of the monomers, a plurality of polymeric
nanoparticles having a core-shell structure may be obtained.

[0064] The first polymer may be a polymer that is able to exhibit an
electric charge, such as a positive charge or a negative charge. The
first polymer may be charged during synthesis of the polymer, and/or in
solution prior to formation of the templates on a surface of the support.
In various embodiments, the first polymer exhibits an electric charge
when present in an aqueous solution. Depending on the type of the first
polymer and/or the pH of the solution, for example, the charge on the
first polymer may be positive or negative. The electric charge on the
first polymer may attract oppositely charged metallic nanoparticles by,
for example, electrostatic attraction. Depending on the type of metallic
nanoparticles for adsorption, and/or the charge type present on the
metallic nanoparticles, different polymers may be used. For example, when
positive charged metallic nanoparticles are used, the first polymer may
be polyacrylic acid, which may acquire a negative charge in an aqueous
medium (at a pH of greater than about 4.5), capable of attracting gold
(Au) nanoparticles functionalized with a positively charged ligand. Other
types of first polymer that exhibit a negative charge in aqueous medium,
which may be dependent on the pH of the solution, may be used.

[0065] As another example, in embodiments in which negatively charged
citrate-stabilized Au nanoparticles are used, the first polymer may be a
polymer that exhibits a positive charge. The first polymer may be a
polymer that exhibits a positive charge in an aqueous medium having a pH
of less than about 8. In various embodiments, the first polymer may
comprise a unit selected from the group consisting of vinyl pyridine,
N-(3-aminopropyl)methacrylamide (APMA),
N-(3-dimethylaminopropyl)methacrylamide, methacrylamidopropyl
trimethylammonium chloride, aminostyrene, ornithine, lysine, amidines,
guanidines, hydrazines, phosphonium salts, and mixtures thereof. In
various embodiments, the first polymer comprises poly(2-vinyl pyridine),
which possesses a vinyl pyridine unit. In some embodiments, the first
polymer is poly(2-vinyl pyridine).

[0066] Generally, the second polymer may comprise any polymer that can
form a shell on the core comprising the first polymer. In some
embodiments, the second polymer comprises a hydrophobic unit. Examples of
such polymers include, but are not limited to, polystyrene, polyolefin,
polysiloxane, polyvinyl naphthalene, polyvinyl anthracene, and mixtures
thereof. In some embodiments, the second polymer comprises polystyrene.
In one embodiment, the second polymer is polystyrene.

[0067] As mentioned above, the first polymer and the second polymer may be
formed independently and processed to form the core and the shell of the
plurality of polymeric nanoparticles, although other techniques may also
be used to form core-shell polymeric nanoparticles. For example, in
various embodiments, the plurality of polymeric nanoparticles may be
formed by copolymerizing the first polymer and the second polymer to form
an amphiphilic copolymer, and dispersing the amphiphilic copolymer in a
suitable organic solvent to form reverse micelles. In some embodiments,
the polymeric nanoparticles are reverse micelles. In these embodiments,
the second polymer may be any suitable polymer that presents a surface
energy contrast with the first polymer to allow formation of micelles in
solution that can be deposited on the support surface to form templates.

[0068] Reverse micelles, also referred to herein as inverted micelles, is
defined as an orientation of amphiphiles in an aggregate structure
whereby the non-polar hydrophobic tails of the amphiphiles are directed
outward into the organic solvent while the polar hydrophilic heads point
inward. As the name implies, in such an orientation, reverse micelles are
opposite to the features of normal micelles in water. Examples of organic
solvents that may be used include, but are not limited to, xylene,
toluene, mesitylene, benzene, pyridine, tetrahydrofuran, pentane, hexane,
heptane, octane, nonane, decane, undecane, dodecane, tridecane,
tetradecane, pentadecane, hexadecane, and mixtures thereof.

[0069] For example, the first polymer and the second polymer may be linked
to form a block copolymer of polystyrene and poly(2-vinylpyridine)
(PS-b-P2VP). When PS-b-P2VP block polymer is dispersed in an organic
solvent, such as m-xylene, the PS-b-P2VP block polymer may form a reverse
micelle in the solvent, whereby the hydrophilic P2VP end of the block
polymer constitutes the core of the reverse micelle, and the hydrophobic
PS end of the block polymer constitutes the outer portion of the reverse
micelle. Accordingly, the polymeric nanoparticles that is used to form
the template may comprise or consist essentially of a block copolymer of
polystyrene and poly(2-vinylpyridine).

[0070] Generally, the reverse micelles formed are spherical in shape
(although other shapes are possible), as such configurations minimize
surface energy. The reverse micelles dispersed in an organic solvent may
be coated on a support using any suitable thin film coating method.
Examples of thin film coating methods include painting, spin coating,
drop coating, tip coating, screen printing and sol gel deposition. In
various embodiments, spin coating is used to form the polymeric
nanoparticles on the support. In embodiments in which the support is an
optical fiber, drop coating may be used.

[0071] Use of an amphiphilic copolymer to form reverse micelles for
nanopattern formation on a support offers advantages such as specific
nanoscale geometry for template size and separation. As mentioned above,
the template size and geometry may be controlled by controlling the size
and geometry of the polymeric nanoparticles by controlling the molecular
weight of the polymer or the polymeric nanoparticle-forming conditions.
For example, the size of the template may be varied by using smaller
micelles, which may be obtained using a copolymer with smaller molecular
weight, micelle formation conditions that result in a smaller aggregation
number, or both. In one embodiment, PS-b-P2VP reverse micelle arrays may
be formed using PS-b-P2VP copolymers having a molecular weight of about
114 kDa. Furthermore, by varying moisture content or humidity in the
surrounding environment during formation of the templates on the support
surface, the curvature of the reverse micelle template on the support may
be tuned in some embodiments due to increases in surface tension at the
interface of the first polymer and the second polymer that constitutes
the reverse micelles. Another advantage of certain embodiments relating
to the formation of templates using reverse micelles lies in that the
reverse micelles formed may be attached in a simple and straightforward
manner using conventional thin film coating methods to generate a
template on a support for subsequent attachment of the metallic
nanoparticles.

[0072] The template of the core-shell polymeric nanoparticles may be
contacted with a plurality of metallic nanoparticles, such that the
metallic nanoparticles attach to the polymeric nanoparticles to form a
metal nanoarray substrate. The nanopatterned polymeric nanoparticles that
are attached to the support may allow for formation of controlled
aggregates of the metallic nanoparticles, which may result in a very low
variation in the surface features of the substrate.

[0073] The polymeric nanoparticles may be irregular or regular in shape.
In some embodiments, the metal nanoparticles are regular in shape. For
example, the polymeric nanoparticles may assume a spherical shape.
Accordingly, the polymeric nanoparticles may be nanospheres.

[0074] The size of the nanoparticles may be characterized by their mean
diameter. The term "diameter" as used herein refers to the maximal length
of a straight line segment passing through the center of a figure and
terminating at the periphery. Accordingly, the term "mean diameter"
refers to an average diameter of the nanoparticles, and may be calculated
by dividing the sum of the diameter of each nanoparticle by the total
number of nanoparticles. Although the term "diameter" is used normally to
refer to the maximal length of a line segment passing through the centre
and connecting two points on the periphery of a nanosphere, it is also
used herein to refer to the maximal length of a line segment passing
through the center and connecting two points on the periphery of
nanoparticles having other shapes, such as a nanocube.

[0075] Although polymeric nanoparticles having a mean diameter in the
sub-micron or micron range may be used, it is advantageous in certain
embodiments for polymeric nanoparticles having a mean diameter in the
nanometer range to be used, in order to generate SERS substrates having a
greater number of nanoparticle cluster features for analysis. The
polymeric nanoparticles may have a mean diameter that is less than 200
nm, such as in the range from about 10 nm to about 100 nm, or about 10 nm
to about 50 nm. In various embodiments, the polymeric nanoparticles have
a mean diameter in the range of about 30 nm to about 200 nm. In one
specific embodiment, the polymeric nanoparticles have a mean diameter of
about 30 nm to about 60 nm, for example about 40 nm.

[0076] Advantageously, the method according to various embodiments may be
used to attach a plurality of polymeric nanoparticles to a support
surface to form an array having an average inter-particle distance of
less than 50 nm on the support, such as less than 40 nm, less than 30 nm,
less than 20 nm or less than 10 nm. In one embodiment, the plurality of
polymeric nanoparticles forms an array having an average inter-particle
distance of about 10 nm on the support.

[0077] The size of the template may be varied by subjecting the polymeric
nanoparticles that are attached to the support surface to a treatment.
Generally, any method that is able to remove at least a portion of the
shell of the polymeric nanoparticles, thereby changing the size of the
template, may be used. In various embodiments, the treatment may also
remove a portion of the core of the polymeric nanoparticles to form
smaller polymeric nanoparticles. In various embodiments, the polymeric
template comprising the polymeric nanoparticles may be removed by the
treatment. The treatment may include any suitable physical or chemical
technique. In some embodiments, the treatment comprises dry etching or
wet etching. Examples of dry etching include, but are not limited to,
plasma etching, sputter etching, and reactive ion etching. In one
embodiment, the treatment comprises reactive ion etching.

[0078] The amount of time for treating the plurality of polymeric
nanoparticles may vary depending on the resolution of the template
required. In embodiments where a non-charged shell is used, the amount of
time for treating the polymeric nanoparticles may be varied to remove a
portion of the non-charged shell in order to allow attachment of the
metallic nanoparticles on the polymeric nanoparticles by electrostatic
interaction. In various embodiments where reactive ion etching is used,
oxygen plasma may be used. The duration of plasma exposure may be in the
range of about 15 s to about 50 s, such as about 20 s to about 40 s,
about 20 s, 30 s, or about 40 s.

[0079] In certain embodiments, such methods include subjecting a plurality
of metallic nanoparticles to the exposed cores of the polymeric
nanoparticles of the template to form the metal nanoarray substrate.

[0080] As mentioned above, the core of the polymeric nanoparticles may
comprise or may be formed from a first polymer exhibiting an electric
charge when present in an aqueous solution. For example, the core of the
polymeric nanoparticles may comprise or may be formed from a first
polymer having an isoelectric point that is higher than the pH of the
solution comprising the metallic nanoparticles. By placing the support in
a solution having a pH that is lower than the isoelectric point of the
first polymer, the polymeric nanoparticles, which are attached to the
support, may attain a positive charge. Subsequently, when metallic
nanoparticles, for example, negatively charged metallic nanoparticles,
are brought into contact with the polymeric nanoparticles, the metallic
nanoparticles may be attracted to the positively charged polymeric
nanoparticles, and may be attached to the polymeric nanoparticles by
electrostatic interaction.

[0081] The term "metallic nanoparticles" refers to a nanoparticle that
comprises a SERS active metal. Examples of a SERS active metal include,
but are not limited to, noble metals such as silver, palladium, gold,
platinum, iridium, osmium, rhodium, ruthenium, copper, and alloys
thereof.

[0082] In some embodiments, the metallic nanoparticles comprises or
consists essentially of one or more noble metals. In one embodiment, the
noble metal is gold. In some embodiments, the metallic nanoparticles
comprise a noble metal. For example, the metallic nanoparticles may have
a core-shell structure, in which the core of the metallic nanoparticles
may be formed from any material such as a polymer or glass, and the shell
of the metallic nanoparticles may comprise or may be formed from one or
more noble metals. In various embodiments, the metallic nanoparticles
comprise or consist essentially of gold. In one specific embodiment, the
metallic nanoparticles are gold nanoparticles.

[0083] The metallic nanoparticles may be present as colloidal metal
nanoparticles in solution. In one specific embodiment, gold nanoparticles
prepared by the Turkevich method, which involves citrate reduction of
chloroauric acid, are used. To inhibit the metallic nanoparticles from
aggregating in solution, negatively charged metallic nanoparticles may be
used. In some embodiments, the negatively charged metallic nanoparticles
are metal nanoparticles carrying a negative charge at the nanoparticle
surface.

[0084] Metallic nanoparticles with a negative surface charge may be
nanoparticles in which the negative charge of the metallic nanoparticles
is conferred by a carboxylic acid, sulfonic acid, carbolic acid or a
mixture of the aforementioned acids which is immobilized at the surface
of the metallic nanoparticles. For example, the carboxylic acid may be,
but is not limited to, citric acid, lactic acid, acetic acid, formic
acid, oxalic acid, uric acid, pyrenedodecanoic acid, mercaptosuccinic
acid, aspartic acid, to name only a few.

[0085] In one specific embodiment, citric acid is used to form negatively
charged gold nanoparticles comprising a surface layer of citrate ions.
For example, the metallic nanoparticles may be citrate-stabilized gold
nanoparticles. Due to the presence of citric acid, the pH of the
colloidal gold nanoparticles solution may be acidic, or weakly acidic.
For example, the pH of the colloidal gold nanoparticles solution may be
less than 7, for example, less than 6.5 or less than 6. In one
embodiment, the pH of the citrate-stabilized gold nanoparticle solution
is about 5.8.

[0086] In various embodiments, the metallic nanoparticles that are
attached to the polymeric nanoparticles of the template are negatively
charged metallic nanoparticles. The metallic nanoparticles may be
attached to the polymeric nanoparticles by electrostatic interaction. The
term "electrostatic interaction" as used herein refers to attraction
between electrically charged molecules, such as between a negatively
charged molecule and a positively charged molecule. The electrostatic
interaction between the metallic nanoparticles and the polymeric
nanoparticles may arise from the positively charged polymeric
nanoparticles that are attached to the support and the negatively charged
metallic nanoparticles, or vice versa.

[0087] As mentioned, the first polymer may have an isoelectric point that
is higher than the pH of the solution comprising the metallic
nanoparticles. The term "isoelectric point" refers generally to the pH of
the solution at which a particular molecule or surface carries no net
electrical charge. Thus, the isoelectric point may refer to the pH of the
solution at which the net charge of the first polymer is zero.
Accordingly, when placed in a solution at which the pH is less than its
isoelectric point, the first polymer may attain a positive charge. Even
though the first polymer may be present in the core of the polymeric
nanoparticles and encapsulated by a non-charged shell comprising a second
polymer, electrostatic attraction may also occur through the non-charged
shell. As mentioned above, an optional treatment may be used to remove a
portion of the non-charged shell, e.g., to allow attachment of the
metallic nanoparticles on the polymeric nanoparticles by electrostatic
interaction. Depending on the type of treatment and the treatment time,
the non-charged shell may be substantially removed to expose the core
comprising the first polymer. In various embodiments, the first polymer
exhibits a positive charge in an aqueous medium having a pH of less than
about 8. In one embodiment, the aqueous medium having a pH of less than
about 8 comprises a gold colloidal solution having a pH of about 5.8.

[0088] The metallic nanoparticles may be irregular or regular in shape. In
some embodiments, the metallic nanoparticles are regular in shape. For
example, the metallic nanoparticles may have a regular shape such as a
sphere, a cube or a tetrahedron. Accordingly, the nanoparticles may be
nanospheres, nanocubes, nanotetrahedra, etc. In some embodiments, the
metallic nanoparticles are spherical in shape. The metallic nanoparticles
may also be of other anisotropic-shaped particles.

[0089] The metallic nanoparticles may have a smaller mean diameter than
that of the polymeric nanoparticles. The metallic nanoparticles may have
a mean diameter of about 2 nm to about 50 nm, such as about 2 nm to about
50 nm, about 2 nm to about 20 nm, about 2 nm to about 10 nm, about 5 nm
to about 10 nm, or about 4 nm to about 6 nm. In some embodiments, the
metallic nanoparticles have a mean diameter of about 10 nm. In one
specific embodiment, the metallic nanoparticles have a mean diameter of
about 5 nm to about 15 nm, for example about 5 nm.

[0090] In some embodiments, the metallic nanoparticles may have the same
shape as the polymeric nanoparticles. For example, the polymeric
nanoparticles and the metallic nanoparticles may both be nanospheres. In
some embodiments, the polymeric nanoparticles and the metallic
nanoparticles have different shapes. For example, the polymeric
nanoparticles may be irregularly shaped and the metallic nanoparticles
may be nanospheres.

[0091] The support may comprise a planar surface onto which the plurality
of polymeric nanoparticles is attached. For example, the support may be
in the form of a flat sheet, or a cuboid, or the planar side of a
hemisphere. The support may also assume other shapes, such as a cylinder,
a sphere, a hemisphere, a pyramid, a diamond, or may be irregularly
shaped. Accordingly, the surface of the support wherein the plurality of
polymeric nanoparticles is attached to may be non-planar. In some
embodiments, the support comprises an optical fiber. In such embodiments,
the polymeric nanoparticles may be attached to the optical fiber by drop
coating. In so doing, an even smaller inter-particle distance or
separation compared to that using spin coating, for example, may be
obtained.

[0092] The plurality of polymeric nanoparticles and/or metallic
nanoparticles may essentially be monodisperse. The term "monodisperse"
refers to nanoparticles having a substantially uniform size and shape. In
some embodiments, the standard deviation of diameter distribution of the
polymeric nanoparticles of the template is equal to or less than 20% of
the mean diameter value, such as equal to or less than 15%, 10%, 5% or 3%
of the mean diameter value. In some embodiments, the diameter of the
polymeric nanoparticles is essentially the same.

[0093] Likewise, the standard deviation of diameter distribution of the
metallic nanoparticles may be equal to or less than 20% of the mean
diameter value, such as equal to or less than 15%, 10%, 5% or 3% of the
mean diameter value. In some embodiments, the diameter of the metallic
nanoparticles is essentially the same.

[0094] The metallic nanoparticles attached to the polymeric nanoparticles
may have an inter-particle distance of less than 5 nm, such as less than
4 nm, less than 3 nm, less than 2 nm or less than 1 nm. The average
number of metallic nanoparticles on each polymeric nanoparticle may be in
the range of about 1 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), and may
depend on the size of the template, for example, the surface area of the
polymeric nanoparticles. Generally, the larger the surface area of the
polymeric nanoparticles, the larger the number of metallic nanoparticles
that may be attached to. In one embodiment, the average number of
metallic nanoparticles on each polymeric nanoparticle is about 18.

[0095] Besides the use of a support having a planar or a non-planar
surface onto which the plurality of polymeric nanoparticles may be
attached, the support may further comprise a metallic nanoparticle
attached to the surface of the support, wherein the metallic nanoparticle
is formed by first forming a polymeric nanoparticle, contacting the
polymeric nanoparticle with a solution containing metal ions, and
removing the polymer, thereby forming metallic nanoparticles in situ. The
term "a metallic nanoparticle" as used herein may refer to one or a
plurality of metallic nanoparticles. Likewise, the term "a polymeric
nanoparticle" as used herein may refer to one or a plurality of polymeric
nanoparticles.

[0096] Examples of polymeric nanoparticles and metallic nanoparticles that
may be used have already been described above. In various embodiments,
the metallic nanoparticle is a gold nanoparticle. In various embodiments,
the polymeric nanoparticle comprises or consists essentially of a block
copolymer of polystyrene and poly(2-vinylpyridine).

[0098] The metal ions may concentrate within the polymeric nanoparticles.
In various embodiments, the metal ions may concentrate within the core
comprising the first polymer of the polymeric nanoparticles.
Subsequently, the polymer may be removed by reactive ion etching. In so
doing, the metal ions that are within the polymeric nanoparticles may
undergo reduction, thereby forming metallic nanoparticles in situ.

[0099] In further embodiments, formation of the template may be carried
out by attaching a plurality of polymeric nanoparticles, some or all
having a core-shell structure, to the metallic nanoparticles that are
attached to the surface of the support. For example, after formation of
the metallic nanoparticles in situ on the support, a plurality of
polymeric nanoparticles, some or all having a core-shell structure such
as that described above, may be attached to the metallic nanoparticles to
form the template.

[0100] In yet further embodiments, formation of the metal nanoarray
includes attaching a plurality of metallic nanoparticles to the polymeric
nanoparticles of the template, and then additional metallic nanoparticles
may optionally be attached to the surface of the support. For example,
after attachment of a plurality of polymeric nanoparticles to the
metallic nanoparticles, which may be formed in situ on the support, a
plurality of metallic nanoparticles may be attached to both the polymeric
nanoparticles and the metallic nanoparticles attached to the surface of
the support. In some embodiments, formation of the template is carried
out by attaching a plurality of polymeric nanoparticles, some or all
having a core-shell structure, directly to the surface of the support.

[0101] In some aspects, the invention refers to a metal nanoarray
substrate obtained by a method such as is described herein.

[0102] In another aspect, the invention refers to a biosensor comprising a
metal nanoarray substrate manufactured by a method such as is described
herein. The biosensor can be configured for in vivo and/or in vitro use.

[0103] In another aspect, the invention refers to a method for the
detection of an analyte in a sample by SERS. The method may comprise
contacting the sample with the biosensor according to aspects such as
those described herein.

[0104] The term "detection" as used herein refers to a method of verifying
the presence of a given molecule. The detection may be qualitative,
and/or the detection may also be quantitative. The detection may include
correlating the detected signal with the amount of analyte. The detection
includes in vitro as well as in vivo detection.

[0105] The term "analyte" as used herein refers to any substance that can
be detected in an assay and which may be present in a sample. The analyte
may, for example, be an antigen, a protein, a polypeptide, a nucleic
acid, a hapten, a carbohydrate, a lipid, a cell or any other of a wide
variety of biological or non-biological molecules, complexes or
combinations thereof. Generally, the analyte will be a protein, peptide,
carbohydrate or lipid derived from a biological source such as bacterial,
fungal, viral, plant or animal samples. Additionally, however, the
analyte may also be a small organic compound such as a drug,
drug-metabolite, dye or other small molecule present in the sample.

[0106] The term "sample", as used herein, refers to an aliquot of
material, frequently biological matrices, an aqueous solution or an
aqueous suspension derived from biological material. Samples to be
assayed for the presence of an analyte by the methods of the present
invention include, for example, cells, tissues, homogenates, lysates,
extracts, and purified or partially purified proteins and other
biological molecules and mixtures thereof.

[0107] Non-limiting examples of samples typically used in the methods of
the invention include human and animal body fluids such as whole blood,
serum, plasma, cerebrospinal fluid, sputum, bronchial washing, bronchial
aspirates, urine, semen, lymph fluids and various external secretions of
the respiratory, intestinal and genitourinary tracts, tears, saliva,
milk, white blood cells, myelomas and the like; biological fluids such as
cell culture supernatants; tissue specimens which may or may not be
fixed; and cell specimens which may or may not be fixed. The samples may
vary based on the assay format and the nature of the tissues, cells,
extracts or other materials, especially biological materials, to be
assayed. For example, methods for preparing protein extracts from cells
or samples are well known in the art and can be readily adapted in order
to obtain a sample that is compatible with the methods of the invention.
Detection in a body fluid can also be in vivo, e.g., without first
collecting a sample.

[0108] By contacting the biosensor with the analyte containing medium, for
example a sample or body fluid, and detecting the SERS signal from the
sensor, the presence of the analyte may be detected. Examples of bodily
fluids that may be used include, but are not limited to, plasma, serum,
blood, lymph, liquor and urine.

[0109] The methods for the detection of an analyte in a sample by SERS may
include, in some embodiments, contacting the sample with one or more
Raman reporters. The term "Raman reporters" refers to compounds which
have a high Raman cross-section and where the Raman vibrational
"fingerprint" is detectably altered, for example by a shift and/or an
increase in intensity, upon the binding of an analyte, so as to allow
detection and/or quantification of the analyte. Accordingly, the
compounds can also be considered to represent reporters or receptors of
the analyte.

[0110] The Raman reporter compounds may bind with the analyte and may be
stably adsorbed at a surface that enhances the Raman signal from the
compounds, such as a substrate, according to various embodiments of the
invention, by reversible electrostatic interaction, hydrophobic
interaction or covalent anchoring. Preferably, the compounds have a high
Raman cross-section and the capability to adsorb strongly on the surface
of the metal nanoparticles so that it gives a fast and intense and non
fluctuating SERS signal that is proportional to the concentration of the
analyte in bulk. Accordingly, by carrying out SERS measurements on the
SERS substrate, the presence and/or quantity of an analyte in a sample
may be determined.

[0111] The use of a biosensor according to various embodiments of the
invention is advantageous in that the SERS-based detection methods of the
invention may be suitable for multiplexing, which is important, for
example, in the context of sensing experiments, to understand complex
mechanistic pathways in biological studies and in personalized medicine.
Furthermore, the use of noble metals, which are biocompatible, in metal
nanoparticles according to various embodiments, means that analyte
detection can be carried out under physiological conditions, and the
sensing components can be integrated in a minimally invasive platform,
such as optical fibers or implantable devices.

[0112] Examples of application areas in which a substrate manufactured by
a method of the invention may be used include, but are not limited to,
analytical devices based on localized surface plasmon resonance (LSPR),
surface enhanced Raman spectroscopy (SERS), metal enhanced fluorescence
(MEF), optical communication devices such as plasmonic waveguides,
lighting devices, solar cells, and photocatalytic devices.

[0113] The inventions illustratively described herein may suitably be
practiced in the absence of any element or elements, limitation or
limitations, not specifically disclosed herein. Thus, for example, the
terms "comprising", "including", "containing", etc. shall be read
expansively and without limitation. Additionally, the terms and
expressions employed herein have been used as terms of description and
not of limitation, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the features shown and
described or portions thereof, but it is recognized that various
modifications are possible within the scope of the invention claimed.
Thus, it should be understood that although various inventions have been
specifically disclosed by preferred embodiments and optional features,
modification and variation of the inventions embodied therein herein
disclosed may be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope of
these inventions.

[0114] Various inventions have been described broadly and generically
herein. Each of the narrower species and subgeneric groupings falling
within the generic disclosure also form part of the inventions. This
includes the generic description of the inventions with a proviso or
negative limitation removing any subject matter from the genus,
regardless of whether or not the excised material is specifically recited
herein.

[0115] Other embodiments are within the following claims and non-limiting
examples. In addition, where features or aspects of the inventions are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.

[0118] The fibers were cut into 10 cm pieces by a cleaver. The jacket and
cladding were stripped to a length of approximately 1.5 cm from each end.
Both ends were then polished using alumina polishing sheets (1 μm)
using standard techniques. The polished ends were then washed for about 2
to 3 minutes with a jet of water and sonicated in ethanol for 10 minutes
and dried.

Example 4

Preparation of Control Substrates (Flats Sheets and Fibers)

[0119] Silicon or glass substrates control substrates with unpatterned
gold nanoparticles were prepared by first treating with UV/Ozone for 10
minutes. Subsequently, the substrates were functionalized with
3-aminopropyl trimethoxy silane in vapor phase, prior to incubation of
the chip in the aqueous solution of citrate stabilized gold
nanoparticles.

[0120] In case of fibers, the cladding was protected with aluminum foil
exposing only the tip to the UV/ozone treatment. Silanization was
performed within a desiccator at a vacuum of 5×10-2 mTorr for
a duration of 2 hours.

Example 5

Deposition of PS-b-P2VP Reverse Micelle Arrays on Substrate

[0121] The silicon and glass substrates were cleaned by ultrasonicating in
acetone followed by 2-propanol and finally treated with UV/ozone. 0.5%
(w/w) of the solution of the polymer was prepared in m-xylene.

[0122] PS-b-P2VP reverse micelle arrays were deposited from 0.5% w/w
solutions of m-Xylene, using polymer with a molecular weight of 114 kDa,
fPS˜0.5 (fPS indicates the volume fraction of the
polystyrene block in the copolymer) and a polydispersity index (PDI) of
1.1. The micelles were deposited onto the substrates in the form of a
thin film by spin coating at 5000 rpm, and an acceleration of 5000 rpm/s
on Si or glass chips to obtain a periodicity of 88 nm. The as-coated
arrays present a two-dimensionally quasi-periodic distribution of P2VP
domains, covered with a thin PS film.

Example 6

Plasma Treatment of PS-b-P2VP Reverse Micelle Arrays on Substrate

[0123] The micellar film was subjected to a controlled exposure to oxygen
plasma to tune the size of the templates. Typically, the duration of
plasma exposure (30 W, 65 mT, 20 sccm) used was 22 s, 30 s and 38 s to
obtain polyelectrolyte template of various sizes.

[0124] Either the as-coated templates or templates with different sizes
obtained after oxygen (O2) plasma exposure were immersed in a
citrate-stabilized gold nanoparticle solution (pH 5.8) for a duration of
2 hours, and this was followed by rinsing in excess deionized water.
Particle clusters were observed to form in 20 minutes, although the 2
hours duration was maintained as a standard for all samples. Longer
durations did not cause a noticeable difference for the cluster
characteristics. In the case of the control substrates, they were
incubated in the aqueous solution of citrate-stabilized gold
nanoparticles for a duration of 12 hours.

[0125] The films were subsequently incubated in an aqueous solution
containing citrate-stabilized gold nanoparticles, with a diameter of 11.6
nm+/-0.79 nm at a pH of 5.8. Spatially selective clustering of
nanoparticles around each micelle feature was observed.

Example 8

Electrostatic Attraction Between P2VP and Gold Nanoparticles

[0126] The P2VP block by the virtue of the basicity of its constituent
pyridyl units exhibits a net positive charge in aqueous medium at mildly
basic to acidic pH values. This is evident from isoelectric point of 8.3
measured using electrokinetic measurements performed on the as-coated
PS-b-P2VP thin films, as seen in FIG. 2, where FIG. 2 is a graph showing
variation in zeta potential of polystyrene-block-poly(2-vinylpyridine)
(PS-b-PVP) thin film with pH of the solution. The isoelectric point (pI)
of 8.3 is indicated. Also indicated is the zeta potential of 30.6 mV at a
pH of 5.8, which is the pH of the citrate stabilized gold nanoparticle
solution.

[0127] The ζ potential of the nanoparticles was electrophoretically
determined to be -38.9 mV. The reverse micelle arrays exhibit a high
positive potential ζ value of 30.6 mV at the pH of the nanoparticle
suspension. Thus, the array of reverse micelles translates into an array
of positively charged centers, consequently guiding negatively charged
gold nanoparticles from solution to adhere strongly to the templates.
This guides the spatially selective clustering of nanoparticles around
each micelle feature.

[0128] Since the P2VP domains are spatially separated from each other, the
clusters obtained are also spatially well isolated from each other. The
number of particles in each such cluster is determined by the surface
area of the reverse micelle template available for the immobilization.

[0129] The size of the template may be varied by using smaller micelles,
which in turn can be obtained either using a copolymer with smaller
molecular weight, or micelle formation conditions that result in a
smaller aggregation number. Alternatively, the as-obtained patterns above
can be exposed to highly controlled oxygen (O2) plasma in a reactive
ion etcher, to systematically etch the polymer in steps of only a few
nanometers. The latter route was used to systematically vary the template
size, while keeping a constant periodicity. The oxygen (O2) plasma
conditions were optimized to have an etch rate of 19.2 nm/min. FIG. 15 is
a graph showing histogram of nanoparticle diameters obtained from
plan-view TEM images, showing average particle size of 11.6 (+/-0.8) nm.

[0131] FIG. 4 provides the histogram of the number of nanoparticles per
cluster (N) obtained as-coated, and using templates obtained upon 20 s,
35 s and 50 s of O2 plasma etching. Clusters of systematically
varying dimensions, viz. N=5, 8, 13 or 18 nanoparticles/cluster and low
standard deviations can be seen from the plan-view TEM images and the
histograms in FIGS. 3 and 4 respectively.

[0132] The SEM analysis (FIG. 18) was used to spot any uncovered areas and
quantify the yield of the cluster formation. Based on this, the yield was
determined to be close to 100%. The defects found on the chip
corresponded only to the occasional presence of dust particles, or edge
defects due to spin-coating process. It should be noted that the clusters
are bound to a template that has a curved geometry. Therefore, the
clusters are not planar and as a consequence the overlapping of the
nanoparticles from different planes of focus can make them appear fused
in some areas of the TEM images.

[0133] The standard deviation observed in the values for N follows
directly from those of the original templates themselves. The templates
exhibit a standard deviation (as percent of mean) of 11.2% for as-coated
templates. As the size of the templates are reduced using O2 plasma
exposures of 22 s, 30 s, and 38 s, the standard deviation increase to
12.6%, 16.5% and 18.8% respectively. These standard deviations are well
within ranges that can be reasonably expected of self-assembled systems,
e.g. copolymers, colloidal spheres etc. reported in literature. Despite
their inherent standard deviations, the arrays were found to exhibit
remarkable uniformity across the coated area and reproducibility across
different batches of preparation. This quality as shown later has very
important implications for the spectroscopic utility of these arrays.

Example 10

Theoretical Calculation of Separation Between Adjacent Particles

[0134] The separation between the adjacent particles is determined by the
repulsive forces caused due to the negatively charged citrate ligands.
The nanoparticles can therefore be considered to exhibit an effective
diameter that is larger than their actual diameters that are measured
using TEM. The TEM in plan-view and cross-section suggests inter-particle
distances are typically less than 5 nm. However, the non-planar 3D
geometry of the clusters can make the sole interpretation of the
separations by TEM to be deceptive. Therefore, a simple model to estimate
the inter-particle distances within the cluster was used. The model is
based on estimating the effective radii of the nanoparticles through a 2D
parking space available per nanoparticle on the template. The available
parking space per particle is calculated by dividing the surface area of
the template by the foot print of the nanoparticles. Assuming a
triangular lattice, the contribution of this parking space would come
from both the difference between effective and real size of the
nanoparticles, as well as the area at the intersection of the features
(FIG. 5). Since the area at the intersections is known through the
packing efficiency (`p`) for a 2D hcp (hexagonal close-packed) lattice,
which is 90.6%, the difference between the effective and real size of the
particles may be calculated:

[0135] Equation (a) provides the surface area of the templates. Equation
(b) provides the foot print area available on the template for each
nanoparticle. reff denotes the effective radius of the
nanoparticles, which is greater than its physical radius r as shown in
the schematic in FIG. 5. Equation (c) takes into account the packing
efficiency in the form of parameter `p` having a value of 90.6% for a
hexagonal close packed (hcp) lattice. The ratio of the surface area
available on a template for the particles to the foot print per particle
provides N (Equation (c)).

[0136] It can be seen that N is a quadratic function of R. Therefore, by
fitting a quadratic function of the form of v=Cx2 to the plot of N
versus R as shown in FIG. 5a, the value of the fitting coefficient C of
0.027 can be used to deduce reff using Equation (d). The
inter-particle separation (s) is twice the difference between the
effective and the actual radii (r) of the particles as shown in Equation
(e) and in FIG. 5b. The goodness of N versus R fit was confirmed with an
adjusted-R2 value of 0.98. The values of reff and s were thus
deduced to be 8.2 nm and 4.6 nm respectively.

[0137] Standard deviation in inter-particle separations can be expected to
be influenced by the size-distribution of the templates itself, which is
about 10% for arrays in their as-coated form. This value for
inter-particle separation between the particles is indicative of the
thickness of the electrical double layer (or, Debye length), which
depends on the ionic strength of the medium.

Example 11

Random Sequential Adsorption (RSA) Based Simulation

[0138] Irreversible adsorption of a monolayer of charged colloids on
oppositely charged surfaces have been studied extensively using random
sequential adsorption (RSA) models. The maximum attainable surface
coverage, also known as the `jamming limit` has been predicted to be
54.7% by RSA based simulations. In this case, the surface coverage of the
nanoparticles on the surface of the polymeric templates can be estimated
to be about 47%. This represents a fairly high coverage, approaching the
jamming limit of RSA.

[0139] In the case of unpatterned polyelectrolyte films, similar surface
coverage has known to take several hours of incubation. Further,
formation of nanoparticle clusters was observed to occur in duration of
30 minutes, suggesting enhanced kinetics of adsorption. The increase in
kinetics is presumably caused due to confinement of adsorption within
nanopatterns. This observation is interesting, considering the fact that
the adsorption of colloidal particles can be considered as a model for
studying the adsorption of biomolecules owing to similarity in sizes.
There is evidence that a confinement of biomolecular immobilization to
nanoscale areas on surface influences both the kinetics of adsorption as
well as their surface density. Influence of nanopatterned areas in
enhancing electrical field intensities during electrodeposition of metal
thin films on surface is also of interest in this context.

[0140] The separation between templates, and consequently that of the
nanoclusters, can be calculated by using experimental values for the
density of micelle templates and their diameter as input.

[0141] The calculation of the separations is carried out in a similar
manner to those between nanoparticles within the cluster adopted in
Equations (a) to (e). The Atemplate represents the actual foot print
area of each template and is obtained using Equation (f). Ateff
represents the available parking area per template as given by Equation
(g), where p has the same connotation as in Equation (c). R' represents
the radius of the imaginary circular area invoked in order to calculate
the separation between the templates. The density (D) of micelle arrays
was found to be 101 features/μm2, when coated at 5000 rpm spin
speed from a 0.5% w/w solution. The separation between the templates
(St) is arrived at using Equations (h) and (i). The separation
between the nanoparticle clusters Sc is then estimated by
subtracting the diameter of the two nanoparticles as shown in Equation
(j) and FIG. 6. The agreement of this model with experiment was tested
for the TEM images for nanoparticle clusters shown in FIG. 3. The
estimated separation of 37 nm agreed well with what is observed in the
plan-view TEM images, confirming the validity of the model.

[0142] The separations between the templates could be varied
systematically by coating the micelle solutions at spin-speeds varying
from 1000 rpm-5000 rpm. AFM images in FIG. 7 show systematic variations
in the densities (and hence, separations) of the templates as well as the
nanoparticle clusters derived from them.

[0143] The separations obtained in each of these cases may be readily
estimated using Equations (f) to (i), with values of 61.0, 53.3, 45.5 and
33.7 nm for the templates, and 37.6, 29.9, 22.1 and 10.3 nm for the
nanoparticle clusters.

[0144] The schematic representation of the templates and the clusters in
top-view and cross-sections before and after immobilization of
nanoparticles is shown in FIGS. 6 (a) and (b). Calculation of edge-edge
separations between the clusters is shown schematically in FIG. 6 (c), to
be derived from the distribution in diameter of the templates, the
clusters, and the periodicity of the array. It should be noted that the
imaging of features that exhibit separations approaching sub-10 nm length
scales are influenced by tip-convolution effects. This arises due to the
steric hindrance felt by the tip in reaching the surface due to its
typical radius of curvatures of about 5 nm to 10 nm. It can be seen as a
consequence that, while the clusters at higher separations may be seen
distinctly, those at the lowest separation show a much lesser distinction
between the individual clusters. Also for the same reason, the AFM images
of the nanoparticles within the clusters look fused, and their
distinction is perceived mainly through their topography.

Example 12

Optical Properties of Metal Nanocluster Arrays

[0145] Aggregation of metal nanoparticles gives rise to unique optical
properties due to electromagnetic multipole interactions present between
the constituent particles. Aggregates of particles are known to absorb
and scatter incident light more efficiently than isolated particles.
Optical properties of regular multi-particle aggregates of metal colloids
that behave like artificial molecules has been reported earlier. The
plasmon coupling between metal nanoparticles has been reported to show
considerable red-shifting of the plasmon resonance. The plasmon coupling
has been shown to be a sensitive function of the inter-particle
separation, and the number of nearest neighbors.

[0146] In case of clusters shown here, the inter-particle separation of
4.6 nm is lower than the particle radius of 5.8 nm, based on which an
excellent coupling is expected. The strong plasmon coupling is reflected
in their extinction spectra (FIG. 8) as a red-shift in resonance peak by
>100 nm as compared to that of isolated nanoparticles. The extinction
spectra show systematic red-shifting of resonance peak position with an
increase in N (FIG. 8b). There is a weak modulation identifiable in the
lower wavelength region between 520 nm to 540 nm, particularly for higher
N values that can be attributed to the isolated particles. In case of
random aggregates of gold nanoparticles reported earlier, the cluster
dimensions as well as inter-particle separations exhibit broad
distributions. This is apparent from their extinction spectra that show
distinctly noticeable peak corresponding to the isolated gold particles
along with a broad peak corresponding to the clusters. This is similar to
those identified in gold nanoparticle heptamers particularly when the
overall cluster size is small. Interesting optical properties such as
Fano resonance have been shown to appear in the spectra of clusters
consisting of larger particles.

[0147] Extinction spectra of nanoparticle clusters show plasmonic peak in
the range of 590 nm to 620 nm corresponding to N=18, but with a
systematic decrease in inter-cluster separations in the sub-50 nm regime.
The extinction spectra show a systematic increase in peak wavelength
until 622 nm.

[0148] The approach shown in this example can be readily extended for
creating clusters with much larger particles, by choosing templates of
correspondingly larger dimensions. The increase in the intensity of the
extinction peaks with increase in N is in accordance with the increase
nanoparticle surface concentrations.

[0149] The homogeneity of the particle clusters obtained may be perceived
from the optical photograph of the samples as shown in FIG. 4b, where
FIG. 4b shows samples of nanoparticle cluster arrays obtained on a glass
chip. As can be seen from the photograph, there is variation in hue
across the samples, which may be attributed to change in cluster size.
Uniformity across the coated area of the chip is readily discernible from
the photograph. This was further confirmed by a low variation in
extinction intensities across the sample.

Example 13

SERS Performance Evaluation of Substrate

[0150] The current fabrication approach utilizes a combination block
copolymer based on templating and nanoparticle self-assembly to create a
two dimensional pattern of clusters. In this case, significant advantage
is derived from the ultra-low separations of <5 nm between particles
within the clusters, with the inter-cluster separations below 50 nm. Such
interacting nanoparticles in a cluster can lead to very high SERS
intensity, and the nature of fabrication may allow for excellent template
uniformity resulting in low point to point variation in intensity of SERS
signals. This is also a significant improvement in terms of ease and
flexibility of fabrication, as the substrates with best SERS enhancements
as shown do not require use of any expensive fabrication equipment, nor a
clean-room environment.

[0151] In order to test the capability of cluster arrays for SERS, SERS
detection using crystal violet (CV) as a model molecule have been carried
out. Crystal violet has been used for the comparison of SERS results by
various other groups in spite of the highly fluctuating nature observed
for the SERS spectrum of the molecule. In fact, the use of a molecule
that is known to exhibit high variation is more suitable to challenge the
substrate performance in terms of analysis of point-to-point variations,
since enhancement factors for a particular substrate in SERS is usually
specific to each molecule and molecular vibrations. Also CV is an
important analyte since there is an illicit use of it in the aquaculture
industry as an antimicrobial in spite of its toxicity and mutagenicity to
mammalian cells.

[0152] Mili-Q water from Elga Purelab Ultra distillation system, having
conductivity of 18.2 MΩ-cm at 26° C., was used in all the
experiments. The SERS experiments were carried out using Raman microscope
(Reinshaw In Via, UK) with an excitation laser at 633 nm. The system is
connected to the microscope (Lecia) and laser light was coupled through
an objective lens of 50× for screening substrate, which was used to
excite the sample, and the return Raman signal would be collected. A
Peltier cooled CCD detector is used by the system to collect all the
Raman signals. Instrument control and data acquisition were taken using
the WIRE 3.0 software package provided with the Renishaw system.
Calibration of instrument was done with the Raman signal from a silicon
standard centered at 520 cm-1.

[0153] SERS substrates were incubated overnight in 1 μM crystal violet
(CV) solution to test for reproducibility and repeatability in SERS
measurement. 12.24 mg of CV powder was weighed and was added to a clean
scintillation vial containing 3 mL of Mili-Q water to make a
concentration of 10 mM CV stock. The mixture was vortexed to ensure
thorough mixing, which vas then followed by wrapping the scintillation
vial with aluminum foil, as CV is light sensitive. The CV stock is stored
in the refrigerator for future use. Dilution was carried out to obtain 5
mL of 1 μM CV solution.

[0154] Substrates in a "wet" condition were obtained from the
scintillation vial containing the CV solution, and fixed onto the glass
slide with a cover slip placed on top of the substrate, and measurements
were done. Substrates in a "dried" condition were obtained from the
scintillation vial of CV solution then put into a beaker of Mili-Q water
and swirled for a few times to wash it. Subsequently, the substrate was
dried with a stream of argon gas and fixed onto the glass slide with a
cover slip placed on top of the substrate, and measurements were done.

[0155] SERS spectra were recorded at 12 random locations on the substrate
using 10 s of exposure time at laser wavelength 633 nm with a power of
6.33 mW using a 20× objective lens. Spectral acquisition was
accomplished using a range of about 400 to 2000 cm-1 with an
exposure time of 10 s and 25% transmission laser power. Subtraction of
baseline was done to eliminate unwanted background noise and to
facilitate data analysis.

[0156] The SERS intensities for the major peaks of CV molecule are
compared amongst arrays with systematically varying cluster size as well
as separations. FIGS. 10 (a) and (c) are graphs showing SERS signal
intensity for the major peaks of CV molecule comparing intensity
enhancement with (a) increases in cluster size N, with N=5, 8, 13 and 18,
and (b) decreases in cluster separation, where separation=37 nm, 30 nm,
22 nm, and 10 nm. From FIGS. 10 (a) and (c), an exponential increase in
the intensity of SERS signals with increases in cluster size from N=5 to
N=18 and decreases in separations from 37 nm to 10 nm may be seen. When
the cluster size was varied, the separation between the clusters was
maintained at a constant value of 37 nm. The highest cluster size of N=18
was maintained throughout when the separations were varied.

[0157] FIGS. 10 (b) and (d) are graphs comparing intensity and
corresponding SERS enhancement factors (EF) of the most intense peak for
CV as a function of (b) cluster size; and (d) separation. The extent of
enhancements can be easily perceived from the plot of signal intensity of
the most intense peak of CV at 1612 cm-1 as a function of cluster
size and separations.

[0158] From the systematic variations in cluster size and separations, a
maximum enhancement was found for the arrays having N=18 clusters with
separation of 10 nm. This substrate was benchmarked against un-patterned
colloidal monolayers consisting of citrate stabilized gold (Au)
nanoparticles adsorbed on amine-terminated self-assembled monolayers of
silane on silicon (Si) substrate.

[0159] As additional comparison, the performance of the substrate obtained
from various embodiments of the invention was compared against that
obtained for commercial Klarite® substrates. FIG. 11 is a bar graph
comparing the signal intensity for the most intense peak of CV molecule
obtained on (b) cluster arrays with N=18 and separation of 10 nm, versus
(a) unpatterned gold nanoparticles on silicon substrates ("unpatterned
control"), and (c) commercial available Klarite® substrates as
controls. The unpatterned control was obtained by adsorption of citrate
stabilized gold nanoparticles on aminosilane treated silicon substrates.
As can be seen from the figure, there is a clear increase in SERS
performance of the clusters as compared with the controls.

[0160] Spectra recorded under the same conditions revealed that the
cluster arrays performed far better in terms of signal intensity and
spectral resolution. The cluster arrays were found to yield peak
intensities that were higher than their unpatterned counterparts by 123%.
The standard deviation in both cases could however be achieved at
<10%, with the clusters exhibiting slightly lower values of 8.5% as
compared to the 9.9% shown by the unpatterned colloid monolayers.

[0161] In order to quantify the actual detection limit possible in cluster
configuration nanoparticles arrays on flat chips, the enhancement factor
for the 1612 cm-1 peak of crystal violet was calculated. The
increase in SERS signal enhancement with increasing cluster size and
decreasing cluster separations as shown in FIGS. 9 and 10 may be related
directly to the hotspot densities. Experimental and theoretical
investigations have shown that maximum enhancement depends on number of
junctions between particles and such junctions are definitely higher in
terms of cluster arrays due to the three dimensional nature of the array
surface compared to the unaltered array. The optimized cluster array
demonstrates a 23% increase in SERS intensity.

Example 14

Characterization of PS-b-P2VP Reverse Micelle Arrays on Optical Fiber

[0162] The cluster arrays were formed on optical fiber platform and a
similar SERS analysis was carried out. In order to quantify the actual
detection limit possible in cluster configuration nanoparticles arrays,
the enhancement factor for the 1612 cm-1 peak of crystal violet was
calculated and found to be higher for cluster arrays by about 136% and
about 636% over unpatterned Au nanoparticle controls, for measurements
performed in remote-sensing and direct configurations, respectively.

[0163] The cluster arrays shown in the previous examples may be used to
obtain high density hotspots uniformly on a fiber faucet. From a
manufacturing point of view, cluster formation through simple
drop-coating of PS-b-PVP reverse micelles at the tip of the optical fiber
followed by self-assembly of gold nanoparticles according to some
embodiments of the invention are deemed very attractive. The significance
of the approach stems from the ease of achieving monolayer coverage
conformally on the tip of an optical fiber faucet without having to
depend on any expensive equipment, nor special clean room or
environmental conditions. Further, the fiber faucet is rough in
topography despite the polishing that they are subjected to. Such
roughness seriously limits capability of most commonly used
nanofabrication approaches in efficiently catering to creating patterns
on an optical fiber tip.

[0164] The facile coverage of the fiber tip by the reverse micelle
templates in a conformal manner was confirmed through AFM imaging as seen
in FIG. 12. FIG. 12 (a) is a tapping mode atomic force microscope (AFM)
image of the template deposited by drop-coating on the tip of a polished
optical fiber. The conformal deposition of the reverse micelles on the
rough asperities of the surface of the fiber tip is clearly discernable.
The scale bar in FIG. 12 (a) denotes a length of 400 nm. FIG. 12 (b) is
an optical photograph of the fiber tip covered with gold nanoparticle
cluster arrays. The scale bar in FIG. 12 (b) denotes a length of 200
μm. FIG. 12 (c) is an optical photograph showing the area where
reflectance spectrum was collected. A microspectrometer measuring a spot
of 77 μm×77 μm was used. The scale bar in FIG. 12 (c) denotes
a length of 100 μm. FIG. 12 (d) is a graph showing the reflectance
spectrum having a plasmonic peak at a wavelength of about 640 nm. Due to
the manner in which the templates are deposited, the templates are
close-packed and exhibit very low separation between adjacent features.
The situation therefore is similar to the smallest separations between
the template features achieved on the flat silicon chip which suggests
that the resulting nanoparticle clusters would exhibit excellent SERS
signal enhancements.

[0165] The fibers coated with the templates were incubated in an aqueous
solution of citrate stabilized gold nanoparticles for duration of 2 h.
The conditions used were maintained the same as for the preparation of
nanoparticle cluster arrays on flat chips. The resulting nanoparticle
arrays were characterized using microspectrometry that allowed acquiring
reflectance measurement on areas measuring only few tens of microns
across. The reflectance spectrum was acquired in spot areas measuring 77
μm×77 μm that reveal a prominent peak at about 640 nm due to
scattering due to localized surface plasmons of the nanoparticle cluster
arrays as can be seen in FIG. 12 (a) to (d).

[0166] The uniformity of the nanoparticle cluster arrays could be
confirmed by the excellent uniformity in peak-wavelength positions for
spectra recorded at different areas of the fiber faucet. The observed
peak wavelength of 640 nm in the case of nanoparticle clusters formed on
the fiber-tip suggests inter-cluster separations that are even lower than
the smallest separations observed on the flat-chips. This enhanced
separation between the features on the fiber tip as compared to the flat
chip can be attributed to the nature of template deposition, where
spin-coating is carried out on flat chips, and drop-coating is carried
out on the optical fiber faucet.

[0167] The drop-coating tends to create close-packed assemblies of reverse
micelles, as supported by the evidence from the atomic force microscope
(AFM) of the templates as shown in FIG. 12. The imaging of the
nanoparticle cluster arrays formed on the fiber faucet by scanning
electron microscopy (SEM) was complicated by the heavy charging of the
non-conducting glass substrate. The AFM measurements, on the other hand,
are significantly limited due to tip-convolution effects for ultra-low
separations as encountered in this case.

[0168] In view of the above, to demonstrate nanoparticle cluster formation
on the fiber, the images obtained from AFM measurements and results from
the microspectrometry measurement performed on the nanoparticle clusters
obtained after self-assembly of gold nanoparticles are used as evidence
for the formation of templates.

Example 15

SERS Performance of Optical Fiber

[0169] The SERS performance of the optical fiber was tested by using CV as
the model analyte as for the flat chips. The configuration used for
measuring the SERS response by the fiber-faucet dipped into the CV
solution is illustrated in FIGS. 13(b) and (c). The fiber end of the
clusters is dipped into a vial containing the CV solution, while the SERS
spectrum of the CV molecule is measured through the other end of the
fiber which faces the objective lens of the Raman spectrometer. For
testing purposes, the Raman spectrum was also measured in backscattering
geometry, under conditions same as that employed on flat-chips.

[0170] A comparison of the remotely performed measurement with direct
measurement in backscattering geometry is shown in FIG. 14. FIG. 14 are
graphs comparing the SERS signal intensity measured under (a) direct
configuration; and (b) indirect configuration for nanoparticle cluster
arrays versus unpatterned controls. The unpatterned control includes
isolated nanoparticles obtained by electrostatic adsorption of gold
nanoparticles to aminosilane treated fiber. The direct measurement
configuration measures SERS under backscattering geometry on the fiber
tip surface incubated in CV solution overnight. The indirect
configuration corresponds to SERS measurement performed through the
fiber, with the cluster containing end dipped in solution and the other
end facing the objective. FIGS. 14 (c) and (d) are graphs comparing
between the most intense peak of CV spectra shown in FIGS. 14 (a) and (b)
respectively.

[0171] In both cases, the SERS performance of the fiber faucet covered
with cluster arrays is compared against control fibers of faucet covered
with randomly adsorbed gold nanoparticles. The control fibers were
prepared in the same way as flat-chip controls, by exposing glass fiber
tips covered with amine terminated silane SAMs to an aqueous solution of
citrate-stabilized gold nanoparticles for duration of 12 h. The direct
SERS measurements performed in the backscattering geometry show that the
signal intensities obtained on the fiber faucet are comparable with that
observed in the case of cluster-arrays with lowest separations on
flat-chips. The unpatterned chips, however, showed a rather lower
response as compared to the flat chips. The remote sensing measurements
clearly reveal both higher signal intensity as well as lower signal
intensity variations for the fiber faucet with clusters as compared to
the control fibers.

[0172] In order to quantify the actual detection limit possible in cluster
fiber configuration nanoparticles arrays, the enhancement factor for the
1612 cm-1 peak of crystal violet was calculated, and found to be
136% and 636% higher for cluster arrays as compared to the unpatterned
nanoparticle controls for signal intensities detected on the indirect or
direct ends of the fiber respectively.

[0173] The copolymer assembly is used to produce two-dimensionally
patterned array of polyelectrolyte centers with sub-100 nm feature
widths, which are then used to create arrays of gold nanoparticle
clusters through simple electrostatic attachment. The spatial
distribution of the polyelectrolyte containing centers can be controlled
in steps of few nanometers to yield separations down to sub-10 nm length
scales. Using this approach, gold nanoparticle clusters with 1<n<20
with 5 nm<δ<40 nm on Si and glass substrates over areas of a
1 cm×1 cm chip have been demonstrated.

Example 16

Comparative Studies Using E-Beam Lithography

[0174] A multi-scale signal enhancement arising out of collective optical
behavior due to the plasmonic coupling between nanoparticles both within
and between the clusters has been investigated. Arrays using e-beam
lithography (EBL) over areas of 25.4 μm×25.4 μm, with average
number of nanoparticles in the clusters (n) and edge-to-edge separation
(δ) in the range of 1<n<20 and 50<δ<1000 nm have
been fabricated. Significantly low inter-cluster separations, and cluster
arrays spanning areas larger than a few tens of microns are not practical
using EBL, due to limitations associated with the use of EBL. Further,
EBL does not efficiently cater to 3D substrates, e.g. glass capillary, or
glass fibers. Further, earlier reports in literature that use gold
colloids to achieve SERS capability often do not quote the inter-particle
separations, despite significant importance of this value toward SERS
enhancements. In addition, when polymeric coatings are used to passivate
the surface of the nanoparticles, it is difficult to achieve ultra-low
separations due to the finite thickness of the passivation layer.

Example 17

Effects of Humidity on Template Formation

[0175] FIG. 28 are graphs showing (a) systematic variation in curvature
(or height (h) to radius (R) ratio) of the reverse micelle templates on
surface, as a function of relative humidity of the environment during
thin film formation; (b) systematic variation in the plasmon resonance of
nanoparticle cluster arrays with variations in the h/R ratio. The
fine-tunability in curvature is a possibility that arises with a
composite core-shell system such as the reverse micelles. The change
arises due to the possible increase in surface tension at the interface
of polystyrene and polyvinyl pyridine, due to absorption of moisture
within PVP, to the extent there is moisture content in the surrounding,
during the formation of templates on surface. Such fine-tunability in
curvature is found to yield tunability in plasmon resonance. It is a
significant capability of interest to achieve higher SERS performance of
the resulting arrays. There is a possibility of fine-tuning the plasmon
resonance close to the molecular absorbance, and the laser excitation
wavelength used in order to realize high SERS enhancements.

[0176] The relative humidity of the environment was controlled between
about 10% to 90%, in a custom-designed environmentally controlled glove
box equipped with a spin-coater inside. It is important to ensure the
relative humidity of the environment at the time of the spin-coating
step, as the influence due to humidity is felt during the template
formation step on surface. The height of the template features for
coatings prepared under different humidity values were characterized
using atomic force microscopy. The templates with different values of
curvature were subjected to incubation in Au nanoparticle solution for a
duration of 3 hours. The optical absorbance of resulting cluster arrays
on glass substrate were measured using a microspectrometer (CRAIC
Technologies). The curvature is presented as a ratio of the height to the
radius of the feature as shown in FIG. 28. The errors due to tip
convolution effects in AFM toward measurement of the radius were taken
into account as indicated by the error bars.

Example 18

Experimental Conditions in Preparation of Super-Clusters

[0177] Polystyrene-block-poly(2-vinylpyridine) with a molecular weight of
380 kDa, with fPS˜0.5 was coated on silicon or glass surface
at 6000 rpm. The template feature exhibited a periodicity (or pitch) of
about 200 nm. The template coated substrate was immersed in 5 mM solution
of chloroauric acid (HAuCl4) for a duration of 1 hour, and then
subjected to O2 plasma RIE for a duration of 10 minutes (at 65
mTorr, 30 W, 20 sccm of O2 flow).

[0178] The gold salt (HAuCl4) concentrates within the PVP blocks of
the template, and the subsequent removal of the polymer therefore
facilitates in the reduction of gold salt and in situ formation of Au
nanoparticles (indicated as `A` in FIG. 29).

[0179] These in situ formed Au particles are subsequently used as
templates for coating reverse micelle templates formed out of PS-b-P2VP
114 kDa and fPS˜0.5. The reverse micelles (indicated as `B` in
FIG. 29) organized around the in situ formed Au NP features. The average
number of reverse micelles per Au nanoparticle (NP) template (indicated
as x) was calculated through histograms, and is as indicated within FIG.
29a. These arrays of super-clusters (ABx) with reverse micelles
surrounding Au NPs were subjected to incubation in a solution of
citrate-stabilized Au nanoparticles as in the earlier examples. The
citrate-stabilized Au nanoparticles (indicated as `C` in FIG. 29a) adsorb
around each reverse micelle feature, and in addition also were found to
adsorb around the central Au nanoparticle template. This results in
super-clusters of nanoparticle cluster arrays (FIG. 29b).

[0180] The adsorption of the `C` to `B` is through electrostatic
attraction (as in the primary examples of nanoparticle cluster formation.
The adsorption of the `C` to `A` however is expected to be mediated
through PS-b-PVP molecules of sub-CMC concentrations that adsorbed on to
`A`, providing it with a positive charge. This aspect is especially
interesting, as it allows the cluster formation to be achieved also
through charged polyelectrolyte functionalization of passive un-charged
nanoparticle surfaces. The composition of the super-clusters of
nanoparticle cluster arrays could be systematically controlled by varying
separation between `A` features, and by controlling the relative humidity
during coating of `B`.

Example 19

Removal of the Polymer Template

[0181] The substrate of the Au nanoparticle cluster arrays was subjected
to O2 plasma reactive ion etching (Oxford plasmlab100, Oxford
Instruments, UK) for a duration of 10 minutes (at 65 mTorr, 30 W, 20 sccm
of O2 flow), to completely remove all the polymeric support. The
characterization of the nanoparticle clusters were performed using atomic
force microscopy and scanning electron microscopy. The arrangement of the
nanoparticle clusters was found to be unaffected. The separation between
individual nanoparticles within the cluster is however not readily known
from this characterization. The SERS analysis of the particle clusters
following the RIE shows significant enhancement over the clusters with
the polymeric support intact. This was found to be the case with both the
nanoparticle clusters, and the nanoparticle super-clusters. This provides
an indirect proof that there the hotspots at the inter-particle junctions
within the clusters are preserved, and that the inter-particle
separations could have decreased as a result of the polymer removal (from
geometric considerations).

Example 20

SERS Enhancement Results

[0182] FIG. 30 are graphs showing removal of supporting polymer template
result in higher SERS enhancements due to a closer separation between
nanoparticles, contrary to earlier beliefs that the nanoparticle formed a
fused mass by coalescing together. All spectra were recorded under
identical conditions of probe molecule deposition, laser excitation
wavelength, exposure duration and laser power. The SERS enhancement was
found to distinctly increase upon removal of the polymer template as
shown in FIG. 30 (a). The enhancement further increased upon formation of
a super-cluster (with polymer removed), as shown in FIG. 30 (b). The
influence on the SERS enhancement due to super-cluster formation may have
contributions from the central gold nanoparticle template, along with the
complex plasmonic coupling due to the super-cluster geometry.

Example 21

Advantages

[0183] A template-driven approach based on copolymers consisting of a
polyelectrolyte block has been developed. In various embodiments, a block
copolymer such as the PS-b-P2VP is used in the form of micelle arrays
deposited on surface to form templates.

[0184] The ability to tune the size and separation of the micelle
templates may be easily and readily translated to control the size and
separation between nanoparticle clusters. As demonstrated herein, the
approach produces particles with sub-5 nm separations, over macroscopic
2D/3D areas, with excellent SERS performance in terms of both signal
intensities as well as reproducibility.

[0185] In addition, since the patterns on surface are formed by deposition
of pre-formed templates from solution-phase, the approach readily caters
to fragile or 3D surfaces or substrates that cannot withstand annealing
at high temperatures. Accordingly, the use of surface-attached templates
using self-assembly approaches, e.g. copolymer lithography using
phase-separated thin films, nanosphere lithography (NSL) or anodic
aluminum oxide (AAO) would therefore be limited in their capability
towards catering to such surfaces.

[0186] The clusters, due to their very low inter-particle separations,
high densities and uniformity, provide excellent opportunity for use as
SERS substrates due to high density of hotspots. In various embodiments,
the inter-particle separations of the metallic nanoparticles may be made
even smaller by formation of `super-clusters` as described above. The
control of inter-particle and inter-cluster distances can lead to
efficient tuning of the SERS properties. Results from the experiments
carried out have shown that the SERS performance of the substrate
increases with increases in cluster size and decreases in inter-cluster
separations. The optimized substrates show excellent SERS performance,
with very low signal variations of ≦10% within and across samples.
The low standard deviations of the polymeric templates employed in the
experiments allow excellent consistency and reproducibility of the SERS
signals. In addition, the best performing SERS substrates shown herein
can be readily realized without the use of expensive equipment of any
kind, on 2D or 3D substrates, and within short times, over arbitrarily
large areas.

[0187] Another important advantage of some embodiments would be the facile
adaptability of fabrication to remote sensing constructs such as optical
fiber probes. This allows easy sampling and avoids the hazards associated
with free space laser beams. Since normal Raman scattering spectroscopy
has been highly successful in remote sensing and biomedical applications,
a lot of effort has been directed at the development of SERS active
optical fibers. It is postulated that formation of a cluster array on
fiber would effectively address the problem of complete blockage of
hollow fibers with nanoparticles that is encountered in many
nanoparticle-based fabrication approaches. Due to the blockage, the
wavelength response of such cluttered fiber probes is very unpredictable
and hence not practical in SERS biosensing. The absence of such
cluttering in the template directed cluster formation in these examples
allow predictable wavelength response and increased collection efficiency
in back scattering geometry, when excitation and collection of signal is
achieved through the fiber using the rear end of the fiber. This type of
bidirectional probes where one side of the fiber features well controlled
cluster arrays and other side used for the input and collection of the
SERS signals would have tremendous practical implication in remote
sampling conditions for SERS based sensing.

[0188] In summary, a simple, yet highly promising means of producing
macroscopic arrays of nanoparticle clusters with engineered SERS response
on both flat and 3D substrates is presented. Nanoparticle clusters may be
efficiently obtained using electrostatic attachment to charged nanoscale
polyelectrolyte template formed out of
polystyrene-block-poly(2-vinylpyridine) copolymer reverse micelles.
Changes in optical properties and the resulting SERS response were
observed by varying the number of nanoparticles per cluster from N=5 to
N=18, and separations of N=18 clusters from 37 nm to 10 nm. The best
signal enhancements were observed for the largest clusters with the
smallest inter-cluster separations. The convenient handles to tune the
size and separation between reverse micelle templates, allow facile
variation in the geometric characteristics of the nanoparticle clusters.
The uniformity and the reproducibility of the templates allowed realizing
clusters that offered both uniform optical properties and reproducibly
low SERS signal intensity variations. Further, such techniques could be
readily translated into a fiber faucet allowing for remote sensing of
analyte by SERS. The characteristics of the templates obtained on the
optical fiber faucet closely resemble those on the flat chip for their
uniformity and observed SERS signal intensities. The SERS performance of
the clusters on flat chips as well as optical fibers was benchmarked
against performance of unpatterned gold nanoparticle monolayers. The
cluster arrays clearly show both higher signal intensity as well as low
signal variations as compared with the controls. The optimization and
further studies on the fabrication side as well as the studies relating
to the fiber configuration and other optical parameters can bring out the
SERS performance to level to cater the ever-increasing need of remote
monitoring of various analyte in biological and chemical sciences.